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Abstract

Islet-grafts can contribute to their own destruction via the elaboration of proinflammatory genes, many of which are transcriptionally regulated by NF-kappaB. Thus NF-kappaB constitutes an enticing gene therapy candidate to improve the success of islet transplantation. To test this hypothesis in vivo, we blocked NF-kappaB in BALB/c (H2(d)) to C57/BL6 (H2(b)) mouse islet allografts by genetically-engineering islets to express the NF-kappaB super-repressor, IkappaBalpha. Here we show by microarray and RTqPCR that islets exhibit an intrinsic early-immediate pro-inflammatory response; with the most highly up-regulated proinflammatory genes comprising the chemokines Cxcl1, Cxcl2, Cxcl10 and Ccl2; the cytokines Tnfalpha and Il6; and the adhesion molecule Icam1. Overexpression of IkappaBalpha inhibited the expression of these genes by 50-95 % in islets and MIN6 beta-cells in vitro, by inhibiting NF-;kappaB-dependent gene transcription. Histological and RTqPCR analysis at post-operative day (POD) 10 revealed that IkappaBalpha transduced islet allografts exhibited improved islet architecture and strong insulin-labelling with decreased Ccl2 and Il6 mRNA levels compared to GFP-transduced control grafts. Despite these protective effects, NF-kappaB blocked islet allografts were promptly rejected in our MHC mismatched mouse model. However, IkappaBalpha expressing grafts did harbour localized 'pockets' of Foxp3(+) mononuclear cells not evident in control grafts. This result suggested to us that the effect of NF-kappaB blockade might synergize with regulatory Tcell sparing Rapamycin. Indeed, combining intragraft IkappaBalpha expression with low-dose Rapamycin increased the mean survival time of islet allografts from 20 to 81 days; with20 % of grafts surviving for greater than 100 days. In conclusion, Rapamycin unmasks the protective potential of intragraft NF-kappaB blockade which can, in some cases, permit permanent allograft survival without continuous systemic immunosuppression.